Epitranscriptomic m5C methylation of SARS-CoV-2 RNA regulates viral replication and the virulence of progeny viruses in the new infection

While the significance of N6-methyladenosine (m6A) in viral regulation has been extensively studied, the functions of 5-methylcytosine (m5C) modification in viral biology remain largely unexplored. In this study, we demonstrate that m5C is more abundant than m6A in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and provide a comprehensive profile of the m5C landscape of SARS-CoV-2 RNA. Knockout of NSUN2 reduces m5C levels in SARS-CoV-2 virion RNA and enhances viral replication. Nsun2 deficiency mice exhibited higher viral burden and more severe lung tissue damages. Combined RNA-Bis-seq and m5C-MeRIP-seq identified the NSUN2-dependent m5C-methylated cytosines across the positive-sense genomic RNA of SARS-CoV-2, and the mutations of these cytosines enhance RNA stability. The progeny SARS-CoV-2 virions from Nsun2 deficiency mice with low levels of m5C modification exhibited a stronger replication ability. Overall, our findings uncover the vital role played by NSUN2-mediated m5C modification during SARS-CoV-2 replication and propose a host antiviral strategy via epitranscriptomic addition of m5C methylation to SARS-CoV-2 RNA.

In this study, we characterized the m 5 C RNA modifications present in SARS-CoV-2 using mass spectrometry (MS) and next-generation sequencing.Our findings revealed that NSUN2, a typical RNA m 5 C methyltransferase, functions as a negative regulator of SARS-CoV-2 replication.We profiled the widespread distribution of m 5 C methylation sites in positive-sense SARS-CoV-2 gRNA.Mechanistically, NSUN2 mediated the methylation of SARS-CoV-2 RNA transcripts and facilitated their degradation.Knockout of NSUN2 notably reduced m 5 C levels in SARS-CoV-2 virion RNA and increased viral infection.By using the transcription and replication-competent SARS-CoV-2 virus-like particles (trVLP) system, we found that the SARS-CoV-2 green fluorescent protein (GFP)/ΔN trVLPs with low NSUN2-mediated m 5 C modification exhibited higher replication ability.Consistently, the progeny SARS-CoV-2 virions from Nsun2 +/− mice (with low m 5 C level) exhibited a stronger replication ability in both Caco-2 cells and K18-hACE2 mice than the progeny SARS-CoV-2 virions from Nsun2 +/+ mice (with normal m 5 C level).Overall, our findings highlight the vital role of NSUN2-mediated m 5 C methylation during SARS-CoV-2 replication and pathogenesis.We outline a paradigm that host has evolved an antiviral strategy via epitranscriptomic addition of m 5 C methylation to SARS-CoV-2 RNA, which will be an insight valuable for developing new anti-coronavirus drugs.

SARS-CoV-2 gRNA contains more abundant m 5 C modification compared to m 6 A and other modifications
To investigate RNA modifications that SARS-CoV-2 RNA contains, we performed liquid chromatography tandem MS (LC-MS/MS) of SARS-CoV-2-infected Caco-2 cells or purified virions (Fig. 1A).We identified several modified ribonucleosides in SARS-CoV-2 RNA, including 2′-O-methylated derivatives of four canonical nucleosides (Am, Cm, Um, and Gm), m 5 C, and m 6 A. Compared to total RNA of uninfected Caco-2 cells (Fig. 1B), m 5 C is the most abundant RNA modification both in total RNA of SARS-CoV-2-infected Caco-2 cells (Fig. 1C) or purified SARS-CoV-2 virion RNA (Fig. 1D) among these modifications.Next, we developed a biosafety level 2 (BSL-2) cell culture system for the production of trVLP, which expresses GFP protein replacing viral N protein (SARS-CoV-2 GFP/ΔN trVLP).We also purified SARS-CoV-2 GFP/ΔN trVLP virion and identified many modified ribonucleosides in its RNA.Consistently, m 5 C is the most abundant modification compared with many other RNA modifications (fig.S1).Furthermore, we performed m 5 C-methylated RNA immunoprecipitation sequencing (m 5 C-MeRIP-seq) in Caco-2 cells, which were infected with SARS-CoV-2 for 48 hours to profile the m 5 C landscape along SARS-CoV-2 transcriptome.As shown in Fig. 1E, the m 5 C is enriched in the region near the 3′ untranslated region.To further investigate whether m 5 C machinery regulates SARS-CoV-2 infection and replication, we knocked down currently known writers or readers (47,48) of mRNA m 5 C modification and NOP2/ SUN RNA methyltransferase family members (49) in Caco-2 cells using small interfering RNAs (siRNAs) and detected the endogenous viral RNA levels of SARS-CoV-2.Knockdown of NSUN2 enhanced endogenous SARS-CoV-2 N and E RNA levels, while the other currently known writers or readers did not (Fig. 2A and fig.S2).Notably, knockdown of METTL3 or METTL14 only mildly enhanced endogenous SARS-CoV-2 N and E RNA levels.We thus speculated that NSUN2 may specifically and notably regulate SARS-CoV-2 replication compared to other currently known methyltransferases or readers.We then constructed NSUN2 knockout cell lines in Caco-2 cells using CRISPR-Cas9 (fig.S3) and found that knockout of NSUN2 showed notable elevation of the RNA levels of SARS-CoV-2 N and E intracellularly and extracellularly (Fig. 2B).Consistently, knockout of NSUN2 dramatically elevated the protein levels of SARS-CoV-2 N (Fig. 2E).
Our previous study identified that NSUN2 participates in the regulation of virus-induced innate immunity responses (50).To block out the effects of interferon, we used ruxolitinib, a potent and selective Janus kinase 1/2 inhibitor that blocks signaling downstream of type I interferon receptors.As shown in Fig. 2C, the promotion of SARS-CoV-2 propagation in NSUN2-knockout cells was not affected by ruxolitinib treatment.This result further confirmed that promotion of SARS-CoV-2 propagation in the NSUN2-deficient cells was independent of the interferon response.Moreover, we also constructed NSUN2 knockout cell lines in Vero E6 cell, which is deficient in interferon secretion, and the same results were also obtained in NSUN2-knockout Vero E6 cell lines compared with wild-type (WT) Vero E6 cells (Fig. 2, D and F).Meanwhile, we observed that the promotion of SARS-CoV-2 propagation in NSUN2-knockout cells was mildly increased when blocking out the effects of interferon, further demonstrating the notable direct NSUN2-mediated regulation of SARS-CoV-2 replication (Fig. 2, B to D).In addition, to explore the global landscape change of sgRNAs after NSUN2 knockout, we verified the presence of sgRNAs in SARS-CoV-2-infected Caco-2 cells using Northern blotting.The result showed that the global landscape of sgRNAs in NSUN2-knockout cells notably increased compared to WT Caco-2 cells (Fig. 2G).The replication of SARS-CoV-2 takes place at endoplasmic reticulum-derived double-membrane vesicles (ER-DMVs).We further checked whether NSUN2 exists in ER-DMVs.We purified the ER-DMVs from SARS-CoV-2-infected cells, and NSUN2 protein was identified in the ER-DMVs (fig.S4A).Moreover, we also perform nuclear and cytoplasmic protein extraction to investigate the subcellular localization of endogenous NSUN2 upon SARS-CoV-2 infection.Consistent with previous results, NSUN2 were detected mostly in the nucleus in uninfected cells.However, upon SARS-CoV-2 infection, the distribution of NSUN2 has changed and is more distributed in the cytoplasm (fig.S4B).These results further demonstrate the colocalization and critical role of NSUN2 in regulation of SARS-CoV-2 replication.
To assess the m 5 C level of SARS-CoV-2 gRNA in virion from WT or NSUN2 knockout cell lines, we further purified SARS-CoV-2 virions released from Vero E6 cell line and Caco-2 cell line and corresponding NSUN2 knockout cell lines.The LC-MS/MS results provided direct evidence of a notable decrease in m 5 C levels in virion RNA after NSUN2 knockout (Fig. 2H).To further confirm the specific m 5 C modification sites, we performed RNA-bisulfite sequencing (RNA-Bis-seq) both in WT Caco-2 cells and NSUN2 knockout Caco-2 cells that were infected with SARS-CoV-2 (Fig. 2I and figs.S5 and S6).We identified a large amount of m 5 C-methylated sites among SARS-CoV-2 gRNA.Here, we showed the unique m 5 C-methylated sites of positive-sense SARS-CoV-2 gRNA in WT groups versus NSUN2 knockout groups (Fig. 2I).These sites were mediated by NSUN2 and scattered across positive-sense gRNA.The relatively high methylation sites were mainly distributed in nsp14, nsp15, nsp16, S, ORF3a, E, M, and N.

The screened m 5 C-methylated cytosines in SARS-CoV-2 were identified to regulate RNA degradation
We then sought to study the function of these relatively high m 5 C methylation sites.We prepared RNA segments of each region of SARS-CoV-2 gRNA by in vitro transcription.The RNAs were used for in vitro methylation assays using recombinant glutathione Stransferase (GST)-NSUN2 and 3 H-labeled S-adenosyl methionine (SAM).These transcribed RNA segments can be methylated by NSUN2 (Fig. 3, A and B).Meanwhile, the data showed that NSUN2 can more efficiently mediate the methylation of N in vitro compared with other regions.Furthermore, we prepared RNA segments with C or m 5 C.These transcribed RNA segments with C or m 5 C were transfected into cells after capping (Fig. 3, A and C, and figs.S7 and S8).The quantitative polymerase chain reaction (qPCR) results in the expression levels of these transcribed RNA segments with m 5 C in nsp14, nsp15, nsp16, S, E, M, and N being lower than those corresponding transcribed RNA segments with C (Fig. 3C and figs.S7 and S8).However, the incorporation of m 5 C mildly promoted the RNA stability of ORF3a but did not affect the RNA degradation of nsp6 (with no notable NSUN2-dependent m 5 C site), indicating that not all m 5 C cause degradation (fig.S7).We speculated that incorporation of m 5 C into these transcribed RNA segments may affect the stability of RNA of nsp14, nsp15, nsp16, S, E, M, and N. We then tested whether these identified highly methylated cytosines were indeed involved in the regulation of mRNA stability.We constructed expression plasmids containing either WT or site mutations (table S2) of methylated cytosines of nsp14, nsp15, nsp16, S, E, M, or N and found that most of the site mutations of methylated cytosines increased the corresponding RNA expression levels (Fig. 3D).We then measured the stability of these transcripts and found that the transcripts of site mutation of methylated cytosines were consistently more stable than the WT transcripts, indicating that methylation of these cytosines by NSUN2 was indeed critical for regulating their mRNA stability and degradation (Fig. 3, E to K, and fig.S9).
Coronaviruses are known to generate sgRNAs through TRSdependent template switching.The N region is located near the 3′ terminus of gRNA.Most of the sgRNAs contain the N region, and the N sgRNA is the most abundant among the various sgRNAs.The results of RNA-Bis-seq (Fig. 2I and fig.S5) and m 5 C-MeRIP-seq (Fig. 1E) both revealed that the N region was highly methylated.Therefore, we chose to further investigate the methylation and biological function of NSUN2 to N. On the one hand, we found that overexpression of NSUN2 inhibited the RNA levels of N, while knockout of NSUN2 elevated the RNA levels of N both in Vero E6 cells and human embryonic kidney (HEK) 293T cells (Fig. 4A and fig.S10A).Reconstitution of WT NSUN2 but not the methyltransferase inactivated mutant (50) in NSUN2 knockout cells restored the

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inhibition function to N RNA levels.Of note, the N transcripts in NSUN2 knockout cells showed higher RNA stability compared to WT cells (Fig. 4B and fig.S10B).Correspondingly, reconstitution of WT NSUN2 but not the methyltransferase inactivated mutant in NSUN2 knockout cells restored the inhibitory function on RNA stability of N transcripts.On the other hand, we developed a BSL-2 cell culture system for production of trVLP, which expresses GFP protein replacing viral N protein (SARS-CoV-2 GFP/ΔN trVLP).We then used the lentiviral system to generate stable WT SARS-CoV-2-N or SARS-CoV-2-N-C28840-mut cell lines in Caco-2 cells, which can be infected by SARS-CoV-2 GFP/ΔN trVLP.By this system, we identified that the SARS-CoV-2 GFP/ΔN trVLP in the SARS-CoV-2-N-C28840-mut Caco-2 cell line showed higher replication level than that in the SARS-CoV-2-N-WT Caco-2 cell line (Fig. 4, C to F).Both intracellular (Fig. 4, C and E) and extracellular viruses (Fig. 4F) were notably elevated in the SARS-CoV-2-N-C28840-mut Caco-2 cell line.Because of the gain of function of SARS-CoV-2 caused by the mutation of the m 5 C methylation sites, reverse genetics cannot be used to make mutant recombinant virus.Together, the results verified that this regulation of N transcripts by NSUN2 was dependent on its m 5 C methyltransferase activity, and the mutation of m 5 C site on N promoted the trVLP replication level.

NSUN2 regulated SARS-CoV-2 replication in two mice models
To further investigate the role of NSUN2 in the regulation of SARS-CoV-2 infection in vivo, we created targeted deletions of NSUN2 in mice by removing 10 base pairs (bps) in exon 3 of Nsun2 genome by CRISPR-Cas9, which resulted in a frameshift mutation (50).However, the homozygous Nsun2 −/− mice died in utero.Therefore, we chose Nsun2 +/− mice as Nsun2 deficiency mice (fig.S11).Then, we used an adenoviral transduction-based mouse model, which can be infected with SARS-CoV-2 in C57BL/6J mice (Fig. 6A) (51).Nsun2 +/+ and Nsun2 +/− mice were anesthetized with isoflurane and intranasally transduced with Ad5-hACE2.Five days after transduction, the mice  were then infected intranasally with SARS-CoV-2.The Nsun2 +/− mice lost more weight than the Nsun2 +/+ mice (Fig. 6B), and there was a higher viral burden of SARS-CoV-2 in lungs of Nsun2 +/− mice than in WT mice at the RNA levels of N and E (Fig. 6C).Meanwhile, Nsun2 +/− mice showed a higher viral burden of SARS-CoV-2 (N protein) in lungs and more severe lung tissue damages compared with their WT littermates (Fig. 6, D and E).
To block the effect of interferon responses induced by NSUN2 deletion, we crossed the Nsun2 +/− mice with Ifnar −/− and got the Ifnar −/− Nsun2 +/+ mice and Ifnar −/− Nsun2 +/− mice.Then, we used SARS-CoV-2 Beta variant (B.1.351)infection mice model to further verify the NSUN2-mediated regulation of SARS-CoV-2 infection in vivo (Fig. 6F).The Ifnar −/− Nsun2 +/− mice lost up to about 10% of their weight in the first 3 to 4 days of infection, while the Ifnar −/− Nsun2 +/+ mice did not show notable weight change (Fig. 6G).Accordingly, Ifnar −/− Nsun2 +/− mice showed a higher viral burden of SARS-CoV-2 in lungs and more severe lung tissue damages compared with Ifnar −/− Nsun2 +/+ mice both at the protein levels and RNA levels (Fig. 6, H to J). Together, the results of the two SARS-CoV-2-infected mice models suggest that NSUN2 was a negative regulator of SARS-CoV-2 and quite pivotal for the replication and pathogenicity of SARS-CoV-2 in vivo, which may serve as a strategy for the host to resist the replication of SARS-CoV-2.

NSUN2 expression was antagonized by SARS-CoV-2 infection
To investigate the biological role of NSUN2 during SARS-CoV-2 infection, we performed RNA sequencing (RNA-seq) to systematically analyze the expression levels of mRNAs in Caco-2 cells or Caco-2 cells with SARS-CoV-2 infection for 24 hours.NSUN2 mRNA levels were consistently decreased in SARS-CoV-2-infected Caco-2 cells compared to mock Caco-2 cells (Fig. 7, A and B).Using the SARS-CoV-2 Beta variant (B.1.351)-infectedmouse model, we found that endogenous Nsun2 mRNA levels also decreased in SARS-CoV-2 Beta variant (B.1.351)-infectedgroup [4 days post infection (dpi)] compared to the uninfected group (Mock) (Fig. 7C).However, there was no notable difference in the endogenous Nsun2 mRNA levels between the infected group (10 dpi) and the uninfected group.According to our results, the SARS-CoV-2 Beta variant (B.1.351)-infectedmouse model showed mild symptoms, and, in the infected group (10 dpi), the mice regained weight and recovered with low viral loads in their lung tissues.These results suggested that when SARS-CoV-2 replicates to a higher level, it suppresses the expression level of NSUN2 through some unknown mechanism.In addition, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) of two patients with COVID-19.NSUN2 mRNA levels were also reduced in patients with COVID-19 compared with healthy individuals (Fig. 7D) (52).From our above results, NSUN2 negatively regulated the replication of SARS-CoV-2 via epitranscriptomic addition of m 5 C modification to SARS-CoV-2 transcripts.This mechanism may be exploited by the host as an antiviral strategy.Therefore, SARS-CoV-2 will antagonize NSUN2 expression via unknown mechanism.We have systematically screened the proteins encoded by SARS-CoV-2 but did not find a specific regulatory factor (fig. S12A).Viral infections will induce the production of interferons and proinflammatory factors as a common feature.Therefore, we speculated whether interferons or proinflammatory factors decreased NSUN2 expression.Then, we screened interferon-β (IFN-β), tumor necrosis factor-α (TNF-α), and interleukin-1β (fig.S12B).However, NSUN2 mRNA levels did not show notable change with treatment of these factors.On the basis of the current data, we cannot give a clear mechanism at this time.We will further explore the specific mechanism of this degradation in follow-up work.The expression levels of the two m 5 C readers, ALYREF and YBX1, also decreased upon SARS-CoV-2 infection, although they were not involved in the regulation of SARS-CoV-2 (Fig. 2A).Still, this result may uncover that SARS-CoV-2 infection will have an impact on host m 5 C machinery and m 5 C methylome.However, the expression levels of m 6 A machinery genes did not show notable or consistent change compared with m 5 C machinery (Fig. 7, B and D).We further collected BALF from patients with severe and mild COVID-19 with the SARS-CoV-2 Omicron subvariant BA.5.2 and found that NSUN2 expression levels and m 5 C levels decreased in patients with severe compared to mild COVID-19 (Fig. 7, E and F).Unfortunately, two of the patients with severe COVID-19 passed away.We then collected the blood of these convalescent patients.The NSUN2 expression levels and m 5 C levels of these convalescent patients restored to the same level as in healthy individuals (Fig. 7, G and H), which is consistent with the above results of SARS-CoV-2 Beta variant (B.1.351)-infectedmouse model (Fig. 7C).These results provide further evidence that m 5 C machinery is involved in the regulatory process of SARS-CoV-2 replication and NSUN2 expression was antagonized by SARS-CoV-2 infection via unknown mechanism.
We further measured different time points to understand the kinetics of m 5 C modification mediated by NSUN2.NSUN2 expression levels gradually decreased upon SARS-CoV-2 infection, while the m 5 C levels of total RNAs gradually increased to the highest value and then decrease (fig.S13).Because NSUN2 is constitutively expressed in uninfected cells, in the early stage of viral infection, the level of NSUN2 is relatively abundant and enough to modify large amounts of the newborn SARS-CoV-2 RNAs.Therefore, the m 5 C levels of total RNAs will gradually increase because of the newborn m 5 C-modified SARS-CoV-2 RNAs.However, in the late stages of viral infection, because of the decrease of NSUN2 caused by SARS-CoV-2 infection, there are fewer NSUN2 proteins in the cell, and large amounts of progeny viruses containing m 5 C modifications are released outside the cell.Then, the m 5 C modification level of total RNAs will begin to decline.

The SARS-CoV-2 virions with low m 5 C modification from Nsun2 +/− mice exhibited a stronger replication ability and caused more severe lung tissue damages in K18-hACE2 mice
To further verify the differences in the replication ability and virulence of SARS-CoV-2 virions with normal or low m 5 C modification in vivo, we used SARS-CoV-2 Beta variant (B.1.351)to infect Nsun2 +/+ mice and Nsun2 +/− mice, respectively, and gently dissociated the lungs to collect the progeny SARS-CoV-2 virions from Nsun2 +/+ mice or Nsun2 +/− mice (Fig. 8A).We quantified the progeny SARS-CoV-2 virions from Nsun2 +/+ mice or Nsun2 +/− mice, analyzed the m 5 C/C ratio using LC-MS/MS of the progeny SARS-CoV-2 virions, and then used them to infect Caco-2 cells and K18-hACE2 mice, respectively.The LC-MS/MS results showed that the m 5 C level of the progeny SARS-CoV-2 virions from Nsun2 +/− mice reduced by about half (Fig. 8B).Consistent with the previous SARS-CoV-2 GFP/ΔN trVLP results in Fig. 5, the progeny SARS-CoV-2 virions from Nsun2 +/− mice (with low m 5 C level) showed a stronger replication ability both in Caco-2 cells (Fig. 8C) and K18-hACE2 mice (Fig. 8D) than the progeny SARS-CoV-2 virions from Nsun2 +/+ mice (with normal m 5 C 10 of 17  level).Meanwhile, the progeny SARS-CoV-2 virions from Nsun2 +/− mice (with low m 5 C level) caused more severe lung tissue damages in K18-hACE2 mice and a higher viral burden of SARS-CoV-2 (N protein) in lungs (Fig. 8, E and F).
Together, these results provide strong evidence that m 5 C modification is an important way to regulate SARS-CoV-2 replication and virulence, and the progeny SARS-CoV-2 virions with low m 5 C modification replicates more strongly inside the host in vivo.NSUN2-mediated m 5 C methylation of SARS-CoV-2 RNA negatively regulates viral replication and the virulence of progeny viruses in the new infection.

DISCUSSION
Inconsistent results of the m 6 A-mediated regulation of SARS-CoV-2 replication have been reported.Two groups reported that m 6 A RNA methylation negatively regulates the SARS-CoV-2 life cycle (36,37), while three groups demonstrated that m 6 A positively regulates SARS-CoV-2 replication (38)(39)(40).We found that knockdown of m 6 A writers mildly increased SARS-CoV-2 replication (Fig. 2A).However, knockdown or knockout of m 5 C writers NSUN2 notably increased SARS-CoV-2 replication.Moreover, according to our results of LC-MS/ MS, m 5 C is more abundant than m 6 A in SARS-CoV-2 virion RNA (Fig. 1D and fig.S1).Together, the above results demonstrate that m 5 C RNA methylation showed more notable regulation of SARS-CoV-2 than m 6 A.
From the results of RNA-Bis-seq and m 5 C-MeRIP-seq (Figs. 1E and 2I and fig.S5), the m 5 C methylation sites were distributed across the entire gRNA, with no particularly hypermethylated m 5 C methylation sites.The Input signal and m 5 C-IP signal near the N region are very high because of the highest expression level of N sgRNAs, but the ratios of m 5 C-IP/Input in N region or other m 5 C-modified regions are close.The N gene contains NSUN2-dependent m 5 C sites, which play notable roles in regulating the function of N expression and the replication of SARS-CoV-2.Meanwhile, the other m 5 C sites were also identified in other regions, such as nsp14, nsp15, nsp16, E, M, and S. The m 5 C incorporation experiment suggested that m 5 C modification had a notable effect on the degradation of viral RNA fragments as a whole effect (Fig. 3C and fig.S8).The identified m 5 C methylation sites among nsp14, nsp15, nsp16, S, E, M, and N were proved to be functional in regulating SARS-CoV-2 RNA stability, and the mutations of these cytosines enhance RNA stability.However, we found that the mutation of the three high methylation sites in ORF3a decreased RNA expression levels, unlike nsp14, nsp15, nsp16, S, E, M, and N. We found that the methylation sites on ORF3a had different regulatory mechanisms from other regions.We are conducting further research to investigate this phenomenon.
Our previous work revealed that NSUN2 serves as a negative regulator of type I interferon responses in antiviral innate immunity during various viral infections (50).Knockout of NSUN2 can enhance type I interferon responses and downstream interferon-stimulated genes (ISGs) expression after some viral infections both in vitro and in vivo, thus inhibiting the replication of vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Sendai virus (SeV).However, the interferon responses caused by SARS-CoV-2 infection is not so strong as VSV, HSV-1, and SeV.SARS-CoV-2 invests substantial resources to block the establishment of the antiviral response (53).Therefore, the enhanced interferon responses by NSUN2 knockout were not so strong in SARS-CoV-2-infected models.Of note, in this work, the regulation of NSUN2 on SARS-CoV-2 is independent of interferon responses but by directly affecting the stability of viral RNAs.This suggests that the direct effect of NSUN2 on SARS-CoV-2 is much greater than the effect on interferon responses during SARS-CoV-2 infection, unlike some other viruses.
The currently known readers of RNA m 5 C modification are not involved in regulating the replication of SARS-CoV-2 (Fig. 2A).The reason may be the irrelevance of their functions.ALYREF was reported to promote the export of m 5 C-modified mRNAs (47).However, SARS-CoV-2 replicates only in the cytoplasm.YBX1 maintains the stability of its target mRNA with m 5 C modification (48), but our results demonstrated that NSUN2 mediated the methylation of SARS-CoV-2 RNA transcripts and facilitated their degradation.These results imply the involvement of unidentified m 5 C reader proteins that may contribute to the regulation of SARS-CoV-2 replication.The specific degradation mechanism induced by m 5 C modification has not yet been clarified clearly and requires further investigation.Another study reported that m 5 C modification of Epstein-Barr virus RNA decreases its stability (46), which may also support the host antiviral strategy we propose via epitranscriptomic addition of m 5 C methylation to viral RNAs.Further work is required to delineate these different mechanisms and roles that different m 5 C readers play in the regulation of various viruses.
Since SARS-CoV-2 itself does not encode the NSUN2 protein, the original SARS-CoV-2 RNA may not contain so many m 5 C modifications.According to our results (Fig. 5), SARS-CoV-2 GFP/ΔN trVLP without NSUN2-mediated m 5 C methylation showed a stronger replication ability compared to SARS-CoV-2 GFP/ΔN trVLP without NSUN2-mediated m 5 C methylation.We propose a SARS-CoV-2-host interaction model.After SARS-CoV-2 infects the host, the gRNA and sgRNAs of SARS-CoV-2 will be modified with m 5 C modification gradually by the host methyltransferase NSUN2.The m 5 C modification can be recognized by the host as a "marker" and degraded by host factors, thus serving as a host antiviral strategy.This marker is quickly recognized and degraded when the progeny viruses with m 5 C modification enters another cell in the new round of infection.From our results (Fig. 7, D to F), the NSUN2 expression level in patients with severe COVID-19 was lower than that in patients with mild COVID-19.Correspondingly, m 5 C modification on viral and host RNA was reduced in patients with severe COVID-19.When the patients recovered, the NSUN2 expression level in patients with COVID-19 returned to normal as in healthy people (Fig. 7, G and H).Because it is not possible to conduct experiments on reinfection of SARS-CoV-2 (with low or normal m 5 C modification) in humans, we used a mouse model to simulate patients with severe and mild COVID-19 and revealed that progeny SARS-CoV-2 virions from Nsun2 +/− mice (with low m 5 C level, simulating patients with severe COVID-19) showed a stronger replication ability and virulence in the new round of infection in a K18-hACE2 mouse model (Fig. 8) than the progeny SARS-CoV-2 virions from Nsun2 +/+ mice (with normal m 5 C level, simulating patients with mild COVID-19 or healthy individuals).The above results may imply that the progeny SARS-CoV-2 viruses transmitted by patients with severe COVID-19 have a stronger replication ability and virulence in the new round of infection when spreading from person to person due to the lack of m 5 C RNA modification (Fig. 9).The mechanism of NSUN2 expression decline caused by SARS-CoV-2 remains to be further investigated, and we are conducting follow-up investigations.
In conclusion, our investigation has revealed a role for NSUN2mediated m 5 C modification in regulating SARS-CoV-2 replication and virulence.We outline a paradigm that the host has evolved an antiviral strategy via epitranscriptomic addition of m 5 C methylation to SARS-CoV-2 RNAs, which will help in the development of anticoronavirus drugs or efficient therapeutic interventions.

SARS-CoV-2 live virus infection assay
All SARS-CoV-2 live virus-related experiments were approved by the level 3 Biosafety Committee (ABSL-3) of Wuhan University.All experiments involving SARS-CoV-2 were performed in the BSL-3 and ABSL-3 facilities of Wuhan University.

Histopathological analysis and immunofluorescence assay for mice tissues
Histopathological analysis and immunofluorescence assay for mice tissues were performed by Wuhan Servicebio Technology Co. Ltd.For histopathological analysis, the mice tissues were immediately fixed in 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E).For immunofluorescence assay, the sections were blocked with phosphatebuffered saline (PBS) containing 3% bovine serum albumin (BSA) at room temperature for 30 min and then incubated with primary antibody (diluted with PBS appropriately) overnight at 4°C.After being washed three times with PBS, the slides were incubated with fluorescent-labeled secondary antibody (appropriately respond to primary antibody in species) at room temperature for 50 min in dark condition.After being washed for another three times with PBS, the slides were incubated with 4′,6-diamidino-2phenylindole (DAPI) solution at room temperature for 10 min in dark condition.

Immunofluorescence assay
Caco-2 or Vero E6 cells were seeded in 48-well plates and infected with SARS-CoV-2 for 24 hours.The cells were inactivated and fixed with 4% PFA at room temperature for 30 min, permeabilized with 0.2% Triton X-100 at room temperature for 10 min, and blocked with 1% BSA at 37°C for 30 min.Then, the cells were incubated with SARS-CoV-2 N antibody (40143-MM05, Sino Biological) diluted in 1% BSA at 37°C for 1 hour, followed by PBS washing.Next, the cells were incubated with Alexa Fluor 594-conjugated goat anti-mouse immunoglobulin G (Thermo Fisher Scientific, A11032) diluted in 1% BSA at room temperature for 30 min, followed by PBS washing.The nucleus was stained with Hoechst 33342 for 5 min.The images were captured with an inverted fluorescence microscope.

RNA isolation and qPCR
Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer's instructions.The isolated RNA was reverse transcribed to cDNA using PrimeScript RT Reagent Kit (Takara, RR037A) or NovoScript Plus All-in-one first-strand cDNA Synthesis SuperMix (Novoprotein).Real-time qPCR was carried out through ABI 7500 Real Time PCR System by SYBR Green Master Mix (YEASEN, 11199ES03) or Taqman Probe Master Mix (YEASEN, 11205ES08).GAPDH was used in normalization via the ΔΔCt method.Primer sequences are shown in table S1.

Liquid chromatography tandem MS
LC-MS/MS was performed by Wuhan Metware Biotechnology Co. Ltd.Briefly, the digestion mixture contains 1 μg of RNA, 1 U of nuclease P1, 10 mM NaCl, and 2 mM ZnCl2 in a final volume of 30 μl.The mixture was incubated at 37°C for 3 hours.Then, 1 U of shrimp alkaline phosphatase and 2.5 μl of ammonium bicarbonate (1 M) were added into the mixture for another 2 hours and diluted to 100 μl.Three microliters of the mixture was injected into the LC-MS/ MS.The nucleosides were separated by a C18 column and detected by triple-quadrupole MS (Shimadzu MS-8050 mass spectrometer, Tokyo, Japan).RNA modifications contents were detected by Wuhan Metware Biotechnology Co. Ltd. (http://metware.cn/)based on the AB Sciex QTRAP 6500 LC-MS/MS platform.

In vitro transcription assays
RNA was in vitro transcribed with CTP or m 5 CTP (Syngenebio, Nanjing, China) as substrates using a MEGAscript T7 Kit (Ambion, USA) according to the manufacturer's instructions.The transcribed RNAs were then capped with the Vaccinia Capping Enzyme (NEB, M2080) and mRNA Cap 2′-O-methyltransferase (NEB, M0366) in the presence of guanosine triphosphate and SAM if the RNAs were used for transfection.

MeRIP-seq
The m 5 C-MeRIP-seq was provided by Cloudseq Biotech Inc. (Shanghai, China).Briefly, m 5 C RNA immunoprecipitation was performed with the GenSeq m 5 C RNA IP Kit (GenSeq, China) by following the manufacturer's instructions.Both the input samples without immunoprecipitation and the m 5 C IP samples were used for RNA-seq library construction with NEBNext Ultra II Directional RNA Library Prep Kit (NEB, USA).The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, USA).Library sequencing was performed on an illumina Novaseq 6000 instrument with 150-bp paired-end reads.The m 5 C-MeRIP-seq data have been deposited in the GSA database under the accession number: HRA002087.

RNA-Bis-seq
The RNA-Bis-seq was provided by Cloudseq Biotech Inc. (Shanghai, China).Briefly, total RNAs for each sample was ribosomal RNA (rRNA)-depleted using GenSeq rRNA Removal Kit (GenSeq Inc.).rRNA-depleted RNA was bisulfite converted and purified using the EZ RNA methylation Kit (Zymo Research).RNA libraries were then constructed with GenSeq Low Input RNA Library Prep Kit (GenSeq Inc.) according to the manufacturer's instructions.The library was quality-controlled with BioAnalyzer 2100 system (Agilent Technologies Inc.) and then sequenced in an Illumina Novaseq 6000 instrument.Analysis of the spike-in showed C to T conversion rates >99%.The RNA-Bis-seq data have been deposited in the GSA database under the accession number (HRA006230).

RNA sequencing
Total RNAs were extracted from indicated cells using TRIzol reagent.DNA digestion was carried out after RNA extraction by deoxyribonuclease I. RNA quality was determined by examining A260/A280 with Nanodrop.RNA Integrity was confirmed by 1.5% agarose gel electrophoresis.Qualified RNAs were finally quantified by Qubit 3.0 with QubitTM RNA Broad Range Assay kit (Life Technologies, Q10210).Two micrograms of total RNAs were used for stranded RNA-seq library preparation using KCTM Stranded mRNA Library Prep Kit (Wuhan Seqhealth Co. Ltd.China, DR08402) for Illumina following the manufacturer's instruction.PCR products corresponding to 200 to 500 bp were enriched, quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.The RNAseq data have been deposited in the GSA database under the accession number (HRA006231).

Preparation of bone marrow-derived dendritic cells and bone marrow-derived macrophages
Bone marrow cells were isolated from C57BL/6J mouse tibia and femur and then cultured for 7 to 9 days in 10% FBS DMEM containing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, 50 ng/ml, Peprotech) for bone marrow-derived dendritic cells or M-CSF (50 ng/ml, Peprotech) for bone marrow-derived macrophages.IFN-β, TNF-α, and IL-1β were from MCE (MedChemExpress) and used at a final concentration of 20 ng/ml.

Constructions
NSUN2 was constructed into the pCAGGS and pGEX6P-1 vector, respectively.SARS-CoV-2 N gene was constructed into the pCAG and pLVX vector, respectively.The other SARS-CoV-2 genes were constructed into the pCAG vector and pCDNA3.1 vector, respectively.The full-length genome of SARS-CoV-2 was divided into seven fragments and inserted respectively into a pUC57 vector.The gRNAs of NSUN2 were constructed into the lentiCRISPR-v2 vector, respectively.

ER-DMVs fractionation
The ER-DMVs purification method was based on a previous article (54) and performed using the Endoplasmic Reticulum Isolation Kit (Sigma-Aldrich, ER0100) according to the manufacturer's instructions.

Construction of the trVLP
The construction method is based on the articles (55,56).In brief, the full-length genome of SARS-CoV-2 was divided into seven fragments and inserted respectively into a pUC57 vector, with a replacement of SARS-CoV-2 N gene by GFP.The seven fragments were amplified using Phanta Super-Fidelity DNA Polymerase (Vazyme, P501-d1).The obtained PCR product was digested by type IIS restriction endonuclease BsaI or BsmBI and then ligated with T4 ligase (NEB) to obtain a full-length cDNA template.The full-length template and N gene were in vitro transcribed and purified, respectively.Then, 20 μg of fulllength mRNA and 10 μg of N mRNA were both added into a 4-mm cuvette (Bio-Rad) containing 0.4 ml of Caco-2-N cells (8 × 10 6 ) in Ingenio Electroporation Solution (Mirus).A single electrical pulse was applied using a GenePulser apparatus (Bio-Rad) set at 270 V, 950 μF.The sample was placed at room temperature for 5 min and then cultured in a 10-cm cell culture dish for 3 days.Then, the supernatant was collected.This trVLP expresses a reporter gene (GFP) replacing SARS-CoV-2 N gene (SARS-CoV-2 GFP/ΔN trVLP).The complete viral life cycle can be achieved and confined in the cells ectopically expressing SARS-CoV-2 N proteins.

Fig. 1 .
Fig. 1.SARS-CoV-2 gRNA contains abundant m 5 C modification.(A) Schematic diagram of lc-MS/MS of purified SARS-cov-2 virions or SARS-cov-2-infected caco-2 cells at multiplicity of infection (MOi) = 0.02 for 48 hour.(B) Analysis of m 5 c/c ratio using lc-MS/MS of total RnAs extracted from uninfected caco-2 cells.(C and D) Analysis of m 5 c/c ratio using lc-MS/MS of total RnAs extracted from SARS-cov-2-infected caco-2 cells (c) or purified SARS-cov-2 virion RnA (d).(E) visualization of m 5 c-MeRiPseq results shows regions of enrichment for m 5 c immunoprecipitation (upper) over input (lower) from SARS-cov-2-infected caco-2 cells.integrative Genomics viewer tracks displaying read distributions from m 5 c-MeRiP-seq (red panel) and input (blue panel) along SARS-cov-2 positive-sense RnA.data are representative of three independent experiments and were analyzed by two-tailed unpaired t test.Graphs show the means ± Sd (n = 3) derived from three independent experiments [or two independent experiments for (e)].

Fig. 2 .
Fig. 2. NSUN2 regulated m 5 C modification and replication of SARS-CoV-2.(A) qPcR analysis of RnA levels of n or e in caco-2 cells transfected with sicontrol or siRnAs for 36 hours, with infection by SARS-cov-2 at an MOi = 0.02 for another 24 hours.(B and C) qPcR analysis of RnA levels of n or e in cell or supernatant from Wt caco-2 cells or nSUn2 knockout cells, with infection by SARS-cov-2 at an MOi = 0.02 for 24 hours, with (B) or without (c) ruxolitinib treatment.(D) qPcR analysis of RnA levels of n or e in cell or supernatant from Wt vero e6 cells or nSUn2 knockout cells, with infection by SARS-cov-2 at an MOi = 0.02 for 24 hours.(E) immunofluorescence analysis of n protein (red) in Wt caco-2 cells or nSUn2 knockout cells, with infection by SARS-cov-2 at an MOi = 0.02 for 24 hours.Scale bar, 50 μm.(F) immunofluorescence analysis of n protein (red) in Wt vero e6 cells or nSUn2 knockout cells, with infection by SARS-cov-2.Scale bar, 50 μm.(G) northern blot results from RnA probes targeting (+) sgRnAs (n probe) in Wt caco-2 cells or nSUn2 knockout cells, with infection by SARS-cov-2.(H) Analysis of m 5 c/c ratio using lc-MS/MS of purified SARS-cov-2 virion RnA from Wt cells or nSUn2 knockout cells.(I) Bis-seq results of unique m 5 c-methylated sites along SARS-cov-2 positive-sense RnA in SARS-cov-2-infected Wt caco-2 cells versus nSUn2 knockout caco-2 cells.data are representative of three independent experiments and were analyzed by two-tailed unpaired t test.Graphs show the means ± Sd (n = 3) derived from three independent experiments [or two independent experiments for (i)].nS, not significant for P > 0.05, **P < 0.01, ***P < 0.001.

Fig. 3 .
Fig. 3. NSUN2 regulated SARS-CoV-2 RNA degradation.(A) Schematic diagram of in vitro-transcribed RnAs with c or m 5 c using in vitro methylation assays or transfection.(B) in vitro m 5 c methylation assays using recombinant GSt-nSUn2 and the in vitro transcripts as indicated.(C) qPcR analysis of RnA levels of the corresponding gene in huh7 cells transfected with transcribed RnAs with c or m 5 c for 10 hours.(D) qPcR analysis of RnA levels of the corresponding gene in huh7 cells transfected with Wt or m 5 c modification site-mutations (mut) of each gene.(E to K) Stability analysis of RnA of each gene in huh7 cells transfected with Wt or m 5 c modification sitemutations (mut) of each gene for 24 hours, with treatment of actinomycin d (Actd) for another 0, 3, 6, and 9 hours.data are representative of three independent experiments and were analyzed by two-tailed unpaired t test.Graphs show the means ± Sd (n = 3) derived from three independent experiments.nS, not significant for P > 0.05, **P < 0.01, ***P < 0.001.

Fig. 9 .
Fig. 9. Schematic diagram of NSUN2-mediated m 5 C modification regulates SARS-CoV-2 replication and the virulence of progeny viruses in the new infection.We used a mouse model to simulate patients with severe and mild cOvid-19 and revealed that progeny SARS-cov-2 virions from Nsun2 +/− mice (with low m 5 c level, simulating patients with severe cOvid-19) showed a stronger replication ability and virulence in the new round of infection in a K18-hAce2 mouse model than the progeny SARS-cov-2 virions from Nsun2 +/+ mice (with normal m 5 c level, simulating patients with mild cOvid-19 or healthy individuals).